Abstract

Publisher Summary An in-depth understanding of the chemical mechanism would include a description of chemical intermediates as well as the structure of the metastable enzyme transition-state complex(es). Kinetic isotope effects provide a uniquely versatile, nonperturbing probe of both kinetic and chemical mechanisms in enzyme-catalyzed reactions. Employing dopamine β-monooxygenase as a model system, this chapter illustrates (1) the determination of the order of substrate addition in multisubstrate enzyme reactions from the variation of isotope effects on Vm/Km for labeled substrate as a function of the concentration of second, unlabeled substrate. (2) The successful measurement of both deuterium and tritium isotope effects on Vm/Km with the requisite accuracy to allow the calculation of an intrinsic isotope effect with an uncertainty of

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